Zymolyase 100t

Manufactured by Nacalai Tesque

Sourced in Japan

Zymolyase 100T is a commercially available enzyme preparation derived from the culture supernatant of Arthrobacter luteus. It is primarily used for the digestion of yeast cell walls, facilitating the release of cellular contents such as proteins and nucleic acids. The core function of Zymolyase 100T is to hydrolyze the β-1,3-glucan component of the yeast cell wall, thereby enabling efficient cell lysis and the extraction of cellular materials for further analysis or experimentation.

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Lab products found in correlation

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20 protocols using zymolyase 100t

Isolation and Characterization of Evolved and Ancestral Yeast Segregants

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The evolved line was mated with the ancestral line, and the resultant diploid cells were induced to sporulate. Sporulated culture was harvested into a microcentrifuge tube and treated with 0.5 mg/ml Zymolyase-100T (Nacalai Tesque, Kyoto, Japan) in 1 M sorbitol at 28 °C for 2 h to remove the ascal wall. Cells were then treated with 2% SDS at 28 °C for 10 min to kill unsporulated diploid cells, before washing with sterile water and vortexing vigorously to attach individual spores to the tube wall. We then added 0.01% Triton X-100 solution to the tube, before vigorous sonication to detach spores from the tube wall and to separate spore clusters. The suspension was diluted and spread on YPD plates to isolate F1 haploid segregants.
We conducted a total of three runs of noise measurement to identify “evolved-like” and “ancestral-like” F1 segregants. The progenies ranking within the top 20% or bottom 20% of the Tdh2-GFP noise level without changing the mean intensity (i.e., within three standard deviations of the control) were selected for the next run of noise measurement. We started with 360 F1 progeny of confirmed ploidy in the first round and obtained 16 “evolved-like” segregants and 20 “ancestral-like” segregants after finishing the third run. These cells were subjected to whole-genome sequencing analysis.

You S.T., Jhou Y.T., Kao C.F, & Leu J.Y. (2019). Experimental evolution reveals a general role for the methyltransferase Hmt1 in noise buffering. PLoS Biology, 17(10), e3000433.

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Cell Fractionation and Mitochondria Isolation

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For cell fractionation, cells exponentially growing in YES were pelleted and resuspended in a buffer containing 0.1 M Tris-HCl (pH 9.3) and 10 mM dithiothreitol. After incubation at 30°C for 30 min, cells were harvested and resuspended in sorbitol buffer (1.2 M sorbitol and 20 mM potassium phosphate buffer, pH 7.5). Spheroplasts were prepared by treatment with Zymolyase-100T (Nacalai Tesque) and lysing enzymes from Trichoderma harzianum (Sigma). Spheroplasts were pelleted, rinsed in sorbitol buffer, resuspended in homogenization buffer (0.6 M sorbitol, 20 mM HEPES-KOH, pH 7.4), and homogenized using a glass homogenizer. Cell debris was removed by centrifugation at 1000 g for 10 min, and the mitochondria-rich fraction was collected by centrifugation at 6500 g for 10 min.

Fukuda T., Ebi Y., Saigusa T., Furukawa K., Yamashita S.I., Inoue K., Kobayashi D., Yoshida Y, & Kanki T. (2020). Atg43 tethers isolation membranes to mitochondria to promote starvation-induced mitophagy in fission yeast. eLife, 9, e61245.

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Immunostaining Yeast Cells Protocols

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Cells were prepared for immunostaining as described (Xaver et al. 2013 (link)) with the following modifications. Cells were fixed with three successive incubations (10–15 min, room temperature) in 3.4% formaldehyde, the latter two in 0.1 M potassium phosphate, 0.5 mM MgCl₂, pH 6.4, and then stored at 4°. Spheroplasting used 0.5 mg/ml Zymolyase 100T (Nacalai USA #07655) in place of Zymolyase 20T. Slides were immunostained overnight at 4° or 4 hr at 30° with a mixture of the two primary antisera diluted in blocking buffer [rat anti-tubulin (ab6160 1:1250; Abcam) and rabbit anti-MYC (sc-789 1:250; Santa Cruz)], washed in PBS (three times, 5 min, room temperature), and then incubated with secondary antisera [Cy3-conjugated donkey anti-rabbit IgG (#711-165-152; Jackson Laboratories) and FITC-conjugated rabbit anti-rat IgG (# F1763; Sigma), both 1:600 in blocking buffer] for 3hr at 30°, followed by three 5-min room temperature washes in PBS. Samples to be examined by DAPI-staining only were treated as described (Goyon and Lichten 1993 (link)) after formaldehyde fixation and storage as above.

Kaur H., GN K, & Lichten M. (2019). Unresolved Recombination Intermediates Cause a RAD9-Dependent Cell Cycle Arrest in Saccharomyces cerevisiae. Genetics, 213(3), 805-818.

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Yeast Spheroplast Formation and Rapamycin Treatment

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Yeast cells grown to mid-log phase were washed with 100 mM Tris-HCl, pH 8.0, containing 10 mM DTT and then converted to spheroplasts by incubation in 0.5× YPD containing 1 M sorbitol and 200 µg/ml zymolyase 100T (Nacalai Tesque) at 30°C for 30 min. Cells were washed with 20 mM HEPES-KOH, pH 7.2, containing 1.2 M sorbitol and incubated in 0.5× YPD containing 1 M sorbitol and 200 ng/ml rapamycin for 3 h. After centrifugation, pelleted cells were resuspended in HSE buffer (20 mM HEPES-KOH, pH 7.2, 1 M sorbitol, and 1 mM EDTA) containing 0.5× Complete protease inhibitor cocktail (Roche) and passed through a polycarbonate membrane filter with a 3-µm pore size (Merck Millipore). The supernatants (lysates) were obtained by removing cell debris by centrifugation and then were treated with 1% Triton X-100 and 100 µg/ml proteinase K on ice for 30 min. Proteolysis was stopped by the addition of 10 mM PMSF, and after trichloroacetic acid precipitation, the proteins were solubilized in urea SDS sample buffer and analyzed by immunoblotting.

Mochida K., Otani T., Katsumata Y., Kirisako H., Kakuta C., Kotani T, & Nakatogawa H. (2022). Atg39 links and deforms the outer and inner nuclear membranes in selective autophagy of the nucleus. The Journal of Cell Biology, 221(2), e202103178.

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Yeast Cell Membrane Fractionation Protocol

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Approximately 1.3 × 10⁸ yeast cells were suspended in 1 ml buffer A (100 mM Tris-HCl [pH 9.4], 40 mM 2-mercaptoethanol) and incubated at room temperature for 10 min. After centrifugation (4 °C, 2000g, 3 min), cells were suspended in 2 ml buffer B (50 mM Tris-HCl [pH 7.5], 1.2 M sorbitol) and incubated with 0.1 mg zymolyase (Zymolyase 100T; Nacalai Tesque) at 30 °C for 30 min to disrupt the cell walls. To remove the zymolyase, the cell suspensions were loaded into 2 ml of 1.8 M sorbitol and centrifuged (4 °C, 2000g, 5 min). Cell pellets were then suspended in lysis buffer (50 mM Hepes/NaOH [pH 6.8], 150 mM NaCl, 10% glycerol, 1 mM DTT, 1 mM PMSF, 1 × protease inhibitor cocktail [Complete EDTA-free, Merck]), lysed by sonication, and centrifuged (4 °C, 2000g, 3 min). The resulting supernatants (total cell lysates) were subjected to ultracentrifugation (4 °C, 100,000g, 30 min), and the pellets (membrane fractions) were suspended in lysis buffer by sonication.

Kato R., Takenaka Y., Ohno Y, & Kihara A. (2024). Catalytic mechanism of trans-2-enoyl-CoA reductases in the fatty acid elongation cycle and its cooperative action with fatty acid elongases. The Journal of Biological Chemistry, 300(2), 105656.

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Subcellular fractionation and protease protection assay

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Cells were grown in YPD overnight to an OD₆₀₀ of ∼2.0. Cells were washed with sterile distilled water twice and transferred to methanol medium. After 4 h of growth at 30°C, cells were harvested by centrifugation at 3000 × g for 10 min and washed with distilled water twice. To create spheroplasts, 0.5 mg/g cells Zymolyase 100T (Nacalai Tesque, San Diego, CA) was added, and cells were incubated for 30 min at 30°C with gentle rotation. Spheroplasts were harvested by centrifugation at 2000 × g for 10 min at 4°C. The pellet was resuspended in homogenization buffer (5 mM 2-(N-morpholino)ethanesulfonic acid/KOH, pH 5.5, 1 M sorbitol, 12.5 μg/ml leupeptin, 5 μg/ml aprotinin, yeast protease inhibitor cocktail, 5 mM NaF). Cells were broken with 20 strokes in a Dounce homogenizer. The homogenate was spun at 2000 × g for 10 min at 4°C to pellet unbroken cells. The postnuclear supernatant was subject to centrifugation for 1 h at 200,000 × g at 4°C. Equivalent volumes of the resulting supernatant (S200) and pellet (P200) were analyzed by Western blot.
Protease protection assays performed on the pellet fraction obtained were done as previously described (Ma et al., 2009 (link)).

Hagstrom D., Ma C., Guha-Polley S, & Subramani S. (2014). The unique degradation pathway of the PTS2 receptor, Pex7, is dependent on the PTS receptor/coreceptor, Pex5 and Pex20. Molecular Biology of the Cell, 25(17), 2634-2643.

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Freeze Fracture Replica Preparation

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A copper EM grid (100 mesh) immersed with yeast or liposome pellets was sandwiched between a 20-µm-thick copper foil and a flat aluminum disc (242; Engineering Office M. Wohlwend) and frozen using an HPM 010 high-pressure freezing machine (Leica). For freeze fracture, the sandwiched sample was transferred to the stage of a Balzers BAF 400 and fractured at −115°C to −105°C under a vacuum of ∼10⁻⁶ mbar. Replicas were made by electron-beam evaporation in three steps: carbon (C; 5 nm in thickness) at an angle of 90° to the fractured surface, platinum-C (2 nm) at an angle of 45°, and C (10 nm) at an angle of 90°. The thickness of evaporation was adjusted by referring to a quartz crystal thickness monitor.
Replicas were treated sequentially with 2.5% SDS in 0.1 M Tris-HCl (pH 8.0) overnight at 60°C, with 0.1 mg/ml Zymolyase 100T (07665–55; Nacalai) in PBS containing 0.1% Triton X-100, 1% BSA (01281–26; Nacalai), and a protease inhibitor cocktail (25955–11; Nacalai) for 2 h at 37°C, and again with 2.5% SDS in PBS overnight at 60°C. In some experiments, the Zymolyase step was replaced with digestion with 0.5% Westase (9005; Takara Bio) in McIlvain citrate phosphate buffer (pH 6.0) containing 10 mM EDTA and 30% FCS (Tsuji et al., 2019 (link)). The replicas were stored in buffered 50% glycerol in PBS at −20°C until use.

Orii M., Tsuji T., Ogasawara Y, & Fujimoto T. (2021). Transmembrane phospholipid translocation mediated by Atg9 is involved in autophagosome formation. The Journal of Cell Biology, 220(3), e202009194.

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Chromosome Spreading and Immunostaining in Yeast

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For chromosome spreading, the yeast cells were first processed into spheroplasts with Zymolyase-100 T (Nacalai Tesque), spread on a clean slide with 1% Lipsol, and fixed with 3% paraformaldehyde containing 3.4% sucrose (6 (link)). For immunostaining, slides with spread nuclei were sequentially incubated with the proper primary and secondary antibodies. Primary antibodies used in this study include mouse monoclonal anti-Myc (sc-40, Santa Cruz Biotechnology), rat monoclonal anti–hemagglutinin (HA; 11867423001, Roche), goat polyclonal anti-Zip1 (sc-48716, Santa Cruz Biotechnology), rabbit polyclonal anti-RPA (AS07214, Agrisera), and rabbit polyclonal anti-GFP (A-11122, Thermo Fisher Scientific). The following secondary antibodies were used in this study: Alexa 488–conjugated donkey anti-mouse/goat/rabbit (A-21202/A-11055/A-21206, respectively; Thermo Fisher Scientific), Alexa 594–conjugated donkey anti-rat/goat (A-21209/A-11058, respectively; Thermo Fisher Scientific). Chromosomal DNA was stained by 4,6-diamidino-2-phenylindole. Fluorescence images were visualized and acquired using a Zeiss fluorescence microscope (Axio Imager.Z2) equipped with an Andor charge-coupled device (CCD) camera (iXon Ultra 897).

Song M., Zhai B., Yang X., Tan T., Wang Y., Yang X., Tan Y., Chu T., Cao Y., Song Y., Wang S, & Zhang L. (2021). Interplay between Pds5 and Rec8 in regulating chromosome axis length and crossover frequency. Science Advances, 7(11), eabe7920.

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Spheroplast Formation from Yeast Cells

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Cell density of 44-hour liquid cultures (YFH1-off and H3^H113N YFH1-off), or exponentially growing cells (WT and H3^H113N), was determined, and equal amounts of cells from each culture were used for producing spheroplasts (2.5 × 10⁹ cells). Cells were washed once with 50 ml of water and once with 400 μl of dithiothreitol (DTT) buffer [100 mM tris-H₂SO₄ (pH 9.2) and 10 mM DTT]. The washed cells were resuspended in 400 μl of DTT buffer and incubated for 20 min at 30°C. Treated cells were then washed twice with 1.3 ml of Zymolyase buffer [20 mM tris-HCl (pH 7.5) and 1.2 M sorbitol], resuspended in 1.3 ml of Zymolyase buffer containing 200 μg of Zymolyase-100T (Nacalai Tesque), and incubated at 30°C for 35 min. Spheroplastization of at least 95% of cells was confirmed under the microscope by a 1:10 dilution into water. Spheroplasts were pelleted at 2000g, and the supernatant was removed.

Campos O.A., Attar N., Cheng C., Vogelauer M., Mallipeddi N.V., Schmollinger S., Matulionis N., Christofk H.R., Merchant S.S, & Kurdistani S.K. (2021). A pathogenic role for histone H3 copper reductase activity in a yeast model of Friedreich’s ataxia. Science Advances, 7(51), eabj9889.

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ATAC-seq of G1-arrested yeast cells

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G1-arrested cells were released in 25 mM HU for 2 h; total 5 × 10⁵ cells were spheroblasted in 400 μl sorbitol buffer (1.4 M sorbitol, 40 mM HEPES-KOH [pH 7.5], 0.5 mM MgCl₂) with 0.5 mg/ml Zymolyase^®-100T (Nacalai Tesque, Inc.) and 10 mM DTT for 15 min at 30 °C prior to incubation with reaction buffer (TD), Tn5 Transposase (TDE1) from Nextera^TM kit and additional 0.01% digitonin in total volume of 50 μl at 37 °C for 30 min. Transposed DNA was purified by MinElute^TM PCR Purification Kit (QIAGEN) and then amplified by PCR with custom primers from Nextera^TM DNA CD Indexes kit. Amplified libraries were then purified by using MinElute^TM PCR Purification Kit (QIAGEN). Paired-end sequencing was completed on the NextSeq^TM 500 (Illumina) with MID output (150 cycles) at the Institute of Molecular Biology, Academia Sinica.

Chong S.Y., Cutler S., Lin J.J., Tsai C.H., Tsai H.K., Biggins S., Tsukiyama T., Lo Y.C, & Kao C.F. (2020). H3K4 methylation at active genes mitigates transcription-replication conflicts during replication stress. Nature Communications, 11, 809.

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